As the majority of infectious microorganisms either colonize or cross mucosal surfaces to enter the host, development of vaccines that induce protective, lasting mucosal immune responses is a crucial public health strategy. dmLT is a promising adjuvant from the heat-labile enterotoxin (LT)-based adjuvant family. It has demonstrated a unique ability to promote mucosal immunity, dose-sparing, and response longevity after parenteral or mucosal immunization in pre-clinical and early phase clinical studies. The interplay between toxicity and adjuvanticity has often complicated a clear perspective on the immunologic and molecular mechanisms occurring with LT-based adjuvants as well as their use clinically. With dmLT?s current safety and efficacy record, it is the best candidate adjuvant from this family. dmLT also provides a unique opportunity to determine the molecular mechanisms of adjuvanticity. As vaccine development moves toward design of more sophisticated, rational, and tailored immunologic responses, understanding the mechanisms of any vaccine component, like dmLT adjuvant, is a primary concern. The objective of this proposal is to define the key molecular mechanisms of dmLT and early biomarkers of adjuvant activity. It has been established that adjuvanticity of LT and dmLT involves activation of cAMP, PKA and the inflammasome in antigen presenting cells (APCs); but no downstream transcriptional effectors, metabolic changes or comprehensive signaling pathway leading to cellular activation have been identified. Our preliminary data indicates that dmLT has multiple signaling responses occurring simultaneously to stimulate antigen-presenting cells to promote vaccine responses. These signaling responses, to which both the A- and B-subunit contribute, are similar to LT in APCs, but not epithelial cells. In the current proposal, we will use our unique arsenal of LT-related proteins, sophisticated omics analyses, and relevant clinical trial samples to definitively answer key mechanistic questions. These include: (1) What are the molecular mechanisms of adjuvanticity of dmLT within antigen presenting cells? (2) Is altered cellular metabolism required for adjuvant stimulation? (3) Using clinical trial samples, does the magnitude of dmLT-induced signaling or serological indicators 24h post-vaccination predict vaccination outcomes? Upon completion of these aims, we expect to generate a much deeper understanding of the major molecular mechanisms of dmLT adjuvant, thereby supporting optimal and rational vaccine development. In addition, we will provide novel information regarding the involvement of host cell metabolism, which has yet to be explored for vaccine adjuvants. This information will further our long-term goal, to identify and utilize the unique properties of dmLT adjuvant within vaccines for the benefit of human health.